(IgA), the main antibody isotype secreted in the gut, shapes the composition of the intestin microbiome via currently unknown mechanisms (5-8). IgA deficiency in mice increases inter-individual variability in the microbiome(9)and decreases diversity (10, 11). The direct effects of igA on bacteria have largely been studied in the context of enteric infection by pathogens(12). However, early studies of IgA in the healthy gut found that the majority of live bacterial cells in feces are bound by IgA(13), reflecting a steady-state IgA response to persistent indigenous microbes(14). Studies show that IgA promotes adherence of commensal bacteria to tissue-cultured intestinal epithelial cells(15, 16), though the in vivo mplications of this observation are unclear. Furthermore, lack of IgA, the most common human immunodeficiency, does not affect lifespan and only modestly increases susceptibility to respiratory and gastrointestinal infections(17), raising the question of why the immune system evolved to invest the considerable energy to produce several grams of IgA daily(18) Bacteroides fragilis is an important member of the human gut microbiome, with beneficial properties that ameliorate inflammatory and behavioral symptoms in preclinical animal models(19-22). This commensal exhibits remarkable single-strain stability(23, 24)and enriched colonization of the gut mucosal surface(25). To explore physical features of B fragilis interaction with the host epithelium, we used transmission electron microscopy (TEM) to visualize colonic tissues of mono-colonized mice. B fragilis commonly formed discrete aggregates of tightly-packed cells on the apical epithelial surface(Fig. 1A)and penetrated the glycocalyx layer of transmembrane mucins, nearly contacting the microvilli 338. IB and fig. S1A and B). Intact B. fragilis cells were also found nestled in the ducts of e crypts of Lieberkuhn(Fig. IC and SIC). We previously identified a genetic locus in B. fragilis, named the commensal colonization factors(ccfABCDE), which is necessary for colonization of colonic crypts(26). To assess how these genes affect bacterial localization to the mucosal surface, we mono-colonized mice with a ccfCDE (Accr mutant By TEM, B fragilis Accf was only found as sparse, individual cells within the epithelial mucosa, excluded from contact with the glycocalyx(Fig. ID and E), and never observed in aggregates as for wild-type bacteria(ig. IF). B. fragilis burden in the colon lumen was identical between strains (fig. S2A), suggesting that the CCF system is required specifically for bacterial aggregation within mucus High-resolution tomograms of bacterial cells in vivo revealed the presence of a thick, fuzzy capsule layer covering wild-type B. fragilis(Fig. IG), which was significantly reduced in B. fragilis Accf(Fig. IH and ID). We sought to investigate the bacterial physiology underlying this ultrastructural change, and potential corresponding effects on colonization. The ccf locus is highly induced during gut colonization(26) and bacterial growth in mucin O glycans(27), indicating the CCF system may sense a specific host-derived glycan. The ccf genes are homologous to polysaccharide utilization systems in which a sigma factor(ccfA) is activated by extracellular glycan sensing(28), thus we hypothesized that ccfA may activate genes involved in mucosal colonization. We overexpressed ccfA in B. fragilis and assessed global gene expression by RNAseq during in vitro growth(without overexpre cf is poorly expressed in culture(26). Of the non-ccfgenes regulated by ccfA, 24 out of 25 genes mapped to the biosynthesis loci for capsular polysaccharides A and C (PSa and Psc) (Fig 2A, 2B and Table S1). Correspondingly, ccf mutation decreased expression of PSc and cience. Author manuscript; available in PMC 2018 November 18(IgA), the main antibody isotype secreted in the gut, shapes the composition of the intestinal microbiome via currently unknown mechanisms (5–8). IgA deficiency in mice increases inter-individual variability in the microbiome (9) and decreases diversity (10, 11). The direct effects of IgA on bacteria have largely been studied in the context of enteric infection by pathogens (12). However, early studies of IgA in the healthy gut found that the majority of live bacterial cells in feces are bound by IgA (13), reflecting a steady-state IgA response to persistent indigenous microbes (14). Studies show that IgA promotes adherence of commensal bacteria to tissue-cultured intestinal epithelial cells (15, 16), though the in vivo implications of this observation are unclear. Furthermore, lack of IgA, the most common human immunodeficiency, does not affect lifespan and only modestly increases susceptibility to respiratory and gastrointestinal infections (17), raising the question of why the immune system evolved to invest the considerable energy to produce several grams of IgA daily (18). Bacteroides fragilis is an important member of the human gut microbiome, with beneficial properties that ameliorate inflammatory and behavioral symptoms in preclinical animal models (19–22). This commensal exhibits remarkable single-strain stability (23, 24) and enriched colonization of the gut mucosal surface (25). To explore physical features of B. fragilis interaction with the host epithelium, we used transmission electron microscopy (TEM) to visualize colonic tissues of mono-colonized mice. B. fragilis commonly formed discrete aggregates of tightly-packed cells on the apical epithelial surface (Fig. 1A) and penetrated the glycocalyx layer of transmembrane mucins, nearly contacting the microvilli (Fig. 1B and fig. S1A and B). Intact B. fragilis cells were also found nestled in the ducts of the crypts of Lieberkühn (Fig. 1C and S1C). We previously identified a genetic locus in B. fragilis, named the commensal colonization factors (ccfABCDE), which is necessary for colonization of colonic crypts (26). To assess how these genes affect bacterial localization to the mucosal surface, we mono-colonized mice with a ccfCDE (Δccf) mutant. By TEM, B. fragilis Δccf was only found as sparse, individual cells within the epithelial mucosa, excluded from contact with the glycocalyx (Fig. 1D and E), and never observed in aggregates as for wild-type bacteria (Fig. 1F). B. fragilis burden in the colon lumen was identical between strains (fig. S2A), suggesting that the CCF system is required specifically for bacterial aggregation within mucus. High-resolution tomograms of bacterial cells in vivo revealed the presence of a thick, fuzzy capsule layer covering wild-type B. fragilis (Fig. 1G), which was significantly reduced in B. fragilis Δccf (Fig. 1H and 1I). We sought to investigate the bacterial physiology underlying this ultrastructural change, and potential corresponding effects on colonization. The ccf locus is highly induced during gut colonization (26) and bacterial growth in mucin O￾glycans (27), indicating the CCF system may sense a specific host-derived glycan. The ccf genes are homologous to polysaccharide utilization systems in which a sigma factor (ccfA) is activated by extracellular glycan sensing (28), thus we hypothesized that ccfA may activate genes involved in mucosal colonization. We overexpressed ccfA in B. fragilis and assessed global gene expression by RNAseq during in vitro growth (without overexpression ccf is poorly expressed in culture (26)). Of the non-ccf genes regulated by ccfA, 24 out of 25 genes mapped to the biosynthesis loci for capsular polysaccharides A and C (PSA and PSC) (Fig. 2A, 2B and Table S1). Correspondingly, ccf mutation decreased expression of PSC and Donaldson et al. Page 2 Science. Author manuscript; available in PMC 2018 November 18. Author Manuscript Author Manuscript Author Manuscript Author Manuscript